Organism: Pseudomonas aeruginosa Etiological Risk Group: Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia
Disease:
Pseudomonas aeruginosa is a bacterium that causes a wide variety of infections in hospital patients and those with impaired immune systems. Infections by this organism are treated with antibiotics; however, with increasing bacterial resistance to antibiotics, this becomes more difficult. In particular, infections by multidrug-resistant strains of P. aeruginosa can be a serious issue as it extremely difficult to find antibiotics that can inhibit the bacteria. The CDC estimates that there are approximately 51,000 cases of infection by P. aeruginosa in the healthcare setting per year with 6,000 of these cases by multidrug-resistant strains. Of the various ways that bacteria can develop resistance to antibiotics, the most common is the development of an enzyme that can neutralize the drug.
In P. aeruginosa, this enzyme is VIM-2, a metallo-beta-lactamase that hydrolyzes the beta-lactam ring in carbapanems to neutralize their effectiveness [1]. As this enzyme is a Class B beta-lactamase, it is able to hydrolyze nearly all beta-lactam antibiotics [2]. This enzyme is also viable for horizontal gene transfer across different bacteria, augmenting the rate at which the bacteria can become multidrug-resistant. Because of the threat that VIM-2 and other metallo-beta-lactamases poses to hospitals and other medical institutions,
Amino Acid Sequence:
Met S P L A F S V D S S G E Y P T V S E I P V G E V R L Y Q I A D G V W S H I A T Q S F D G A V Y P S N G L I V R D G D E L L L I D T A W G A K N T A A L L A E I E K Q I G L P V T R A V S T H F H D D R V G G V D V L R A A G V A T Y A S P S T R R L A E V E G N E I P T H S L E G L S S S G D A V R F G P V E L F Y P G A A H S T D N L V V Y V P S A S V L Y G G C A I Y E L S R T S A G N V A D A D L A E W P T S I E R I Q Q H Y P E A Q F V I P G H G L P G G L D L L K H T T N V V K A H T N R S V V E Length: Amino Acid Sequence with His Tag:
M H H H H H H S S G V D L G T E N L Y F Q S M S P L A F S V D S S G E Y P T V S E I P V G E V R L Y Q I A D G V W S H I A T Q S F D G A V Y P S N G L I V R D G D E L L L I D T A W G A K N T A A L L A E I E K Q I G L P V T R A V S T H F H D D R V G G V D V L R A A G V A T Y A S P S T R R L A E V E G N E I P T H S L E G L S S S G D A V R F G P V E L F Y P G A A H S T D N L V V Y V P S A S V L Y G G C A I Y E L S R T S A G N V A D A D L A E W P T S I E R I Q Q H Y P E A Q F V I P G H G L P G G L D L L K H T T N V V K A H T N R S V V E Length: 269 aa Weight: 28.802 kDa Molar Extinction Coefficient: 31400 pI: 5.15
Figure 3. TmPred prediction of transmembrane region. There is one potential transmembrane region, but considering this the role of VIM-2, this is unlikely an actual transmembrane region.
DNA Sequence: ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAACGCAGTCGTTTGATG GCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAGATTGAGAAGCAAATTGGACTTCCTGTAAC GCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCGTCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACG CACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTGCTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTG GTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTGGCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGG CCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTGTAAAAGCGCACACAAATCGCTCAGTCGTTGAGTAG Sequence with His Tag: ATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCG GGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAACGCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGC GTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAGATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGG GTGGCAACGTACGCATCACCGTCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATC CTGGTGCTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTGGCTGAATG GCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTGTAAAAGCGCACACAAATCGCTCA GTCGTTGAGTAG Length: 744 bp GC Content: 56.048387% CDS Codon Optimized: N/A Overlap Primers: N/A
Gene ID: 14678525
Protein ID: YP_007509601
Organism: Pseudomonas aeruginosa
Etiological Risk Group: Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia
Disease:Pseudomonas aeruginosa is a bacterium that causes a wide variety of infections in hospital patients and those with impaired immune systems. Infections by this organism are treated with antibiotics; however, with increasing bacterial resistance to antibiotics, this becomes more difficult. In particular, infections by multidrug-resistant strains of P. aeruginosa can be a serious issue as it extremely difficult to find antibiotics that can inhibit the bacteria. The CDC estimates that there are approximately 51,000 cases of infection by P. aeruginosa in the healthcare setting per year with 6,000 of these cases by multidrug-resistant strains. Of the various ways that bacteria can develop resistance to antibiotics, the most common is the development of an enzyme that can neutralize the drug.
In P. aeruginosa, this enzyme is VIM-2, a metallo-beta-lactamase that hydrolyzes the beta-lactam ring in carbapanems to neutralize their effectiveness [1]. As this enzyme is a Class B beta-lactamase, it is able to hydrolyze nearly all beta-lactam antibiotics [2]. This enzyme is also viable for horizontal gene transfer across different bacteria, augmenting the rate at which the bacteria can become multidrug-resistant. Because of the threat that VIM-2 and other metallo-beta-lactamases poses to hospitals and other medical institutions,
TDR_Targets: None
GeneDatabase: http://www.ncbi.nlm.nih.gov/gene/14678525
Essentiality: Essential for neutralizing carbapenams and other beta-lactamases.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC89788/
Druggability:
BindingDB shows one current inhibitor with Ki value:
http://bindingdb.org/jsp/dbsearch/PrimarySearch_ki.jsp?tag=pol&submit=Search&target=beta-lactamase%20vim-2&polymerid=50004301
E.C.: 3.5.2.6
Structure: 1 polymer, 1 or 2 chains
PDB: 1KO2, 1KO3, 4BZ3, 4C1D, 4C1E, 4NQ2, 4PVO, 4PVT, 4UA4
Reaction: Beta-lactam + water -> substituted beta-amino acid
http://www.brenda-enzymes.org/enzyme.php?ecno=3.5.2.6&Suchword=&organism%5B%5D=Pseudomonas+aeruginosa&show_tm=0
Cloning:
DH5a E. coli, pNIC-Bsa4 vector, Kanamycin and Sucrose selection system
Results:
6 positive clones out of 8 tested colonies.
Colonies 1, 2, 4, 5, 6, 8
ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAA
CGCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGA
GATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCG
TCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTG
CTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCT
GGCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTT
GTAAAAGCGCACACAAATCGCTCAGTCGTTGAGTAG
Colony 3
ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAA
CGCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGA
GATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCG
TCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTG
CTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCT
GGCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTT
GTAAAAGCGCACACNAATCGCTCAGTCGTTGAGTAG
Colony 7
ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATTGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAAC
GCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAG
ATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCGT
CGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTGC
TGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTG
GCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTG
TAAAAGCGCACACAAATCGCTCAGTCGTTGAGTAG
Expression:
BL21(DE3) E. coli, induced by IPTG
Results:
Successful transformation into BL21(DE3) cells.
Purification:
6x His Tag, Ni-NTA column purification with imidazole
FPLC
Results:
(gel here)
Enzyme Assay:
Colorimetric assay using Nitrocefin as substrate.
Substrate:
Nitrocefin
http://www.toku-e.com/product/nitrocefin/
Current Inhibitors:
Sodium 2,3-dimethylmaleate
Amino Acid Sequence:
Met S P L A F S V D S S G E Y P T V S E I P V G E V R L Y Q I A D G V W S H I A T Q S F D G A V Y P S N G L I V R D G D E L L L I D T A W G A K N T A A L L A E I E K Q I G L P V T R A V S T H F H D D R V G G V D V L R A A G V A T Y A S P S T R R L A E V E G N E I P T H S L E G L S S S G D A V R F G P V E L F Y P G A A H S T D N L V V Y V P S A S V L Y G G C A I Y E L S R T S A G N V A D A D L A E W P T S I E R I Q Q H Y P E A Q F V I P G H G L P G G L D L L K H T T N V V K A H T N R S V V E
Length:
Amino Acid Sequence with His Tag:
M H H H H H H S S G V D L G T E N L Y F Q S M S P L A F S V D S S G E Y P T V S E I P V G E V R L Y Q I A D G V W S H I A T Q S F D G A V Y P S N G L I V R D G D E L L L I D T A W G A K N T A A L L A E I E K Q I G L P V T R A V S T H F H D D R V G G V D V L R A A G V A T Y A S P S T R R L A E V E G N E I P T H S L E G L S S S G D A V R F G P V E L F Y P G A A H S T D N L V V Y V P S A S V L Y G G C A I Y E L S R T S A G N V A D A D L A E W P T S I E R I Q Q H Y P E A Q F V I P G H G L P G G L D L L K H T T N V V K A H T N R S V V E
Length: 269 aa
Weight: 28.802 kDa
Molar Extinction Coefficient: 31400
pI: 5.15
ProtParam:
http://web.expasy.org/cgi-bin/protparam/protparam
TmPred:
DNA Sequence:
ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAACGCAGTCGTTTGATG GCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAGATTGAGAAGCAAATTGGACTTCCTGTAAC GCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCGTCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACG CACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTGCTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTG GTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTGGCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGG CCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTGTAAAAGCGCACACAAATCGCTCAGTCGTTGAGTAG
Sequence with His Tag:
ATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCG GGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAACGCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGC GTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAGATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGG GTGGCAACGTACGCATCACCGTCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATC CTGGTGCTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTGGCTGAATG GCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTGTAAAAGCGCACACAAATCGCTCA GTCGTTGAGTAG
Length: 744 bp
GC Content: 56.048387%
CDS Codon Optimized: N/A
Overlap Primers: N/A
Primers (pLIC Cloning):
Forward: 5’ TACTTCCAATCCATGAGTCCGCTCGCTTTTTCC 3’
Reverse: 5’ TATCCACCTTTACTGCTACTCAACGACTGAGCGAT 3’